Orchestration of the cell division cycle includes a series of checkpoints which ensure that some events are completed before others begin (Murray, A. et al. The Cell Cycle Oxford University Press (1993). One set of controls determines whether cells replicate their genome in preparation for division (G1/S), while another checks that DNA replication is complete and that the cell has grown sufficiently for division to take place (G2/M) (Nasmyth, K. Science 274:1643-1645 (1996). Cyclins and cyclin-dependent kinases (CDKs) regulate these events in part by controlling the transcription of specific effector genes (Okayama, H. et al., Adv. Cancer Res. 69:17-62 (1996); Sanchez, I. et al., Curr. Opin. Cell. Biol. 8:318-824 (1996)). In budding yeast, CDC28 regulates the transcription of genes whose products are needed for the G1/S transition or S phase (Andrews, B. J. et al., J. Biol. Chem. 265:14057-14060 (1990); Johnston, L. H. et al., Nucl. Acids Res. 20:2403-2010 (1992)) via the transcription factors SBF (Swi4-Swi6) and DSC1 (Swi6-Mpb1) (Andrews, B. J. et al., Cell 57:21-29 (1989); Dirick, L. et al., Nature 357:508-513 (1992); Lowndes, N. F. et al., Nature 357:505-508 (1992); Lowndes, N. F. et al., Nature 350:247-250 (1991); Taba, M. R. et al., Genes Dev. 5:2000-2013 (1991)). In fission yeast, the SBF-related heterodimer, MBF, is required for the expression of similar genes (Aves, S. J. et al., EMBO J. 4:457-463 (1985); Lowndes, N. F. et al., Nature 357:505-508 (1992); Tanaka, K. et al., EMBO J. 11:4923-4932 (1992)). In mammalian cells, the CDK-regulated transcription factor E2F plays a key role in regulating the G1/S transition (Muller, R., Trends Genet. 11:173-178 (1995); Sanchez, I et al., Curr. Opin, Cell. Biol. 8:318-824 (1996)). E2F is complexed with Rb until Rb phosphorylation by G1 cyclin-dependent kinases releases E2F to activate transcription of immediate early genes including myc, fos, and jun (Beijersbergen, R. L. et al., Biochim. Biophys. Acta 1287:103-120 (1996)).
Similar cyclin-dependent control mechanisms regulate the G2/M transition (Forsberg, S. L. et al., Annu. Rev. Cell. Biol. 7:227-256 (1991); Nurse, P., Cell 79:547-550 (1994); Nurse, P., Nature 344:503-508 (1990)), but less is known about their downstream targets (Stukenberg, P. T. et al., Curr. Biol. 7:338-348 (1997)). In fission yeast, regulation of the Cdc2-Cdc13 cyclin-dependent kinase-cyclin complex by the Weel kinase and Cdc25 phosphatase is thought to be the primary mechanism controlling G2/M (Okayama, H. et al., Adv. Cancer Res. 69:17-62 (1996); Russell, P. et al., Cell 49:559-567 (1987)). The Cdc2-Cdc13 complex accumulates during S phase, but Cdc2 is phosphorylated and thereby maintained in an inactive state by Weel (Fleig, U. N. et al., Semin. Cell. Biol. 2:195-205 (1991); Lundgren, K. et al., Cell 65:1111-1122 (1991)). As cells complete DNA replication, Weel is phosphorylated by Nim1 and thereby inactivated (Russell, P. et al., Cell 49:559-567 (1987)), and Cdc25 accumulation leads to the dephosphorylation of Cdc2 (Gautier, J. et al., Cell 67:197-211 (1991); Moreno, S. et al., Nature 344:549-552 (1990)). Dephosphorylation and activation of Cdc2 heralds progression through G2 and entry into mitosis. The biochemical events controlling G2/M transit in mammalian cells are remarkably similar to those in S. pombe. Mammalian Cdc2 kinase accumulates in S phase (Shimizu, M. et al., Mol. Cell. Biol. 15:2882-2892 (1995)) and is regulated by a Weel kinase (Parker, L. L. et al., Science 257:1955-1957 (1992)) and Cdc25 phosphatase (Honda, R. et al., FEBS Lett. 318:331-334 (1993)). While G2/M progression requires the coordinated expression of many genes, how this is regulated at the level of transcription remains largely unknown. The identification and characterization of transcription factors regulating G2 progression and mitotic entry, therefore, would significantly advance our understanding of the mechanisms controlling this portion of the cell cycle.
Most mammalian cells, such as hepatocytes, reside in G0 and can re-enter the cell cycle and undergo mitosis. Significant exceptions to this general rule include skeletal and cardiac myocytes, which are terminally differentiated and apparently incapable of undergoing mitosis shortly after the postnatal period. Tam et al. disclosed the possibility that reversal of terminal differentiation in cardiac myocytes might be achieved by manipulation of pocket proteins and/or cyclin D and cdk2 expression and function (Annals NY Acad Sci. 752: 72-79 (1995). Kirshenbaum et al. (J. Biol. Chem. 270:7791-7794 (1995)) disclosed the reactivation of DNA synthesis, but not proliferation, of cardiac myocytes by the adenoviral protein E1A in concert with E1B delivered via an adenovirus vector. Kirshenbaum et al. (Dev. Biol. 179:402-411 (1996)) disclosed that E2F-1 delivered via an adenovirus vector together with E1B can also activate DNA synthesis and cause the accumulation of cardiac myocytes in G2/M.
S. pombe cdc5p was first described as a putative DNA binding protein implicated in G2/M transit Nasmyth, K. et al. (1981) Mol Gen Genet 182, 119-24). We subsequently identified a cDNA encoding a protein with limited homology to S. pombe cdc5p (Berstein, H. S. et al. (1997) J. Biol. Chem. 272, 5833-7). Its widespread expression in human tissues and homology with expressed sequences in other eukaryotes suggested an evolutionarily conserved general function (Bernstein, H. S. et al. (1997) J. Biol. Chem. 272, 5833-7). Nuclear import upon serum stimulation of mammalian cells suggested a possible role in cell proliferation (Bernstein, H. S. et al. (1997) J. Biol. Chem. 272, 5833-7).
Effector genes regulated by other members of the Cdc5 family in S. cerevisiae (Ohi, R. et al. (1998) Mol Cell Biol 18, 4097-108), A. thaliana (Hirayama, T. et al (1996) Proc Natl Acad Sci USA 93, 13371-6), C. elegans (Bernstein, H. S. et al (1997) J Biol Chem. 272, 5833-7), D. melanogaster (Katzen, A. L. et al. (1998) Genes Dev 12, 831-43; Ohi, R. et al (1998) Mol Cell Biol 18, 4097-108), and M. musculus (Bernstein, H. S. et al. (1997) J Biol Chem 272, 5833-7) have not been identified. Cdc5-related proteins contain tandem helix-turn-helix DNA binding motifs at their amino termini, similar to that seen in Myb-related proteins (Bernstein, H. S. et al. (1997) J Biol Chem 272, 5833-7). In contrast with c-Myb, however, Cdc5-related proteins contain only two repeats of the helix-turn-helix motif, whereas Myb family members possess three (Bernstein, H. S. et al. (1997) J Biol Chem 272, 5833-7; Hirayama, T. et al. (1996) Proc Natl Acad Sci USA 93, 13371-6; Katzen, A. L. et al. (1998) Genes Dev 12, 831-43; Ohi, R et al. (1998) Mol Cell Biol 18, 4097-108; Ohi, R. et al. (1994) EMBO J 13, 471-83). Moreover, within this domain Cdc5-related proteins bear a Valxe2x86x92Leu substitution at a position critical for DNA binding specificity (Carr, M. D. et al. (1996) Eur J Biochem 235, 721-35; Ogata, K. et al. (1996) Nat Struct Biol 3, 178-87). Cdc5 family members, therefore, likely differ from Myb in their DNA binding properties.
Recently a 7 bp nucleotide sequence identified by random oligonucleotide binding site selection was shown to interact with the DNA binding domain of A. thaliana in vitro, however, binding with this sequence was reduced with non-specific competitor DNA (Hirayama, T. et al. (1996) Proc Natl Acad Sci USA 93, 13371-6). Similar experiments to identify a consensus binding site for the highly conserved DNA binding domains of D. melanogaster Cdc5 and the Cdc5 homologue in S. cerevisiae, Cef1p, failed to identify any preferential site, nor did they interact with the 7 bp sequence identified for A. thaliana (Ohi, R. et al. (1998) Mol Cell Biol 18, 4097-108). In addition, others have shown that Cdc5 does not activate the transcription of candidate genes known to be upregulated during G2/M, for example cdc2 and String in D. melanogaster (Katzen, A. L. et al. (1998) Genes Dev 12, 831-43), Clb1 and Swi5 in S. cerevisiae (Ohi, R. et al. (1998) Mol Cell Biol 18, 4097-108), and cdc13+ and cdc25+ in S. pombe (Ohi, R. et al. (1998) Mol Cell Biol 18, 4097-108). These results raised the question of whether Cdc5 family members serve functions other than as site-specific DNA binding proteins (Ohi, R. et al. (1998) Mol Cell Biol 18, 4097-108), even though they possess a Myb-like DNA binding domain. In addition, recent studies in fission and budding yeast have implicated a role for cdc5p and Cef1p, respectively, in pre-mRNA splicing (McDonald, W. H. et al. (1999) Mol Cell Biol 19, 5352-62; Tsai, W. Y. et al. (1999) J Biol Chem 274, 9455-62), and hCdc5 has been identified as a component of the mammalian splicesome (Burns, C. G. et al. (1999) Proc Natl Acad Sci USA 96, 13789-13794; Neubauer, G. et al. (1998) Nature Genet 20, 46-50).
One aspect of the invention is an isolated nucleic acid having the sequence of FIG. 2D (SEQ ID NO: 11).
A further aspect of the invention is an antisense nucleic acid comprising a nucleic acid sequence complementary to the nucleic acid sequence of FIG. 2D (SEQ ID NO: 11).
A further aspect of the invention is a method for treating a cell cycle defect in patient comprising administering to cells in the patient a therapeutic amount of an hCdc5 protein.
A further aspect of the invention is a method for treating a cell cycle defect in a patient comprising administering to cells in the patient an antagonist of hCdc5.
A further aspect of the invention is a method of treating a patient having a hyperproliferative disease, comprising administering to hyperproliferative cells in the patient nucleic acid comprising an hCdc5 nucleic acid, wherein the polypeptide encoded by the nucleic acid is over-expressed, and as a result of the over-expressed polypeptide the cells die.
A further aspect of the invention is a method of regulating the progression of a cell cycle through G2 and into mitosis, comprising administering to a cell an antagonist of hCdc5.
A further aspect of the invention is a polypeptide having the amino acid sequence of FIG. 1A (SEQ ID NO:1).
A further aspect of the invention is a hCdc5 binding site nucleic acid and vectors comprising a hCdc5 binding site nucleic acid. In particular embodiments, the vector comprises the hCdc5 binding site nucleic acid operably linked to nucleic acid encoding a protein of interest Also included is a method of using the vector for expressing the protein of interest in a cell in which hCdc5 is expressed and a method of detecting the expression of hCdc5 in a cell.
Another aspect of the invention is a method for identifying a compound that acts as an effector of the interaction between hCdc5 and the hCdc5 DNA binding site. The effector compound can be an inhibitor or an enhancer of the interaction. Inhibitors identified by the method are useful for treating a patient having a hyperproliferative disease. Enhancers identified by the method are useful for promoting cell division in normally non-regenerable cells. The combination of hCdc5 protein and its DNA binding site nucleic acid is also useful for controlling the regulation of a recombinant gene.